The mitochondrial inner membrane is composed of polypeptides which enter it from both matrix and cytoplasmic (i.e. intermembrane space) surfaces. Those polypeptides which enter the membrane from the matrix side are encoded by mitochondrial genes, translated on ribosomes which are bound to the matrix face of the inner membrane, and inserted into the membrane co-translationally. Those polypeptides which enter the cytoplasmic face are encoded by nuclear genes, translated by soluble ribosomes (or, in some cases, ribosomes bound to the outer mitochondrial membrane), and inserted into the membrane post-translationally. Both classes of membrane proteins must be correctly targeted to the inner membrane and then localized to the oligomeric protein complex within which they reside. In this study we propose to identify the molecular determinants which operate to target and sort one representative yeast polypeptide of each class to a protein oligomer within the inner membrane. We will use yeast cytochrome c oxidase subunit II, a mitochondrial gene product, and yeast cytochrome c oxidase subunit V, a nuclear gene product, as the representative polypeptides. For subunit V, we plan to identify, by directed mutagenesis and intragenic revertant analysis, those domains within the polypeptide and its "leader peptide" which are required for its functional insertion into the inner membrane. We also plan to identify, by the analysis of extragenic revertants, genes which specify ancillary proteins (soluble and membrane) that are required for its insertion into the inner membrane. For subunit II, we plan to analyze the function of its "leader peptide" and, through revertant analysis, identify genes whose protein products interact with its "leader peptide." Finally, we hope to determine if the insertion of these two proteins into opposite sides of the inner membrane can occur simultaneously. This study should enhance our understanding of membrane protein biogenesis in general and the biogenesis of the inner mitochondrial membrane, an essential component of cellular respiration, in particular.